1. Introduction 211

2. Screening methods 213

2.1 Chemical shifts 213

2.2 Diffusion 214

2.3 Transverse relaxation 218

2.4 Nuclear Overhauser effects 218

3. Strategies for drug discovery and design 221

3.1 Fragment-based methods 221

3.1.1 Linked-fragment approach 221

3.1.2 Directed combinatorial libraries 222

3.1.3 Modification of high-affinity ligands 223

3.1.4 Solvent mapping techniques 223

3.2 High-throughput NMR-based screening 224

3.3 Enzymatic assays 226

4. Discovery of novel ligands 227

4.1 High-affinity ligands for FKBP 227

4.2 Potent inhibitors of stromelysin 229

4.3 Ligands for the DNA-binding domain of the E2 protein 233

4.4 Discovery of Erm methyltransferase inhibitors 233

4.5 Phosphotyrosine mimetics for SH2 domains 236

5. Conclusions 237

6. References 237

A critical step in the drug discovery process is the identification of high-affinity ligands for macromolecular targets. Traditionally, the identification of such lead compounds has been accomplished through the high-throughout screening (HTS) of corporate compound repositories. Conventional HTS methodology has enjoyed widespread application and success in the pharmaceutical industry and, through recent technological advances in screening (Fernandes, 1998; Oldenburg et al. 1998; Silverman et al. 1998) and combinatorial chemistry (Fauchere et al. 1998; Fecik et al. 1998), these programs will continue to have a prominent role in drug discovery. However, suitable leads cannot always be found using conventional methods. This is not surprising since typical corporate libraries contain fewer than 106 compounds compared with the estimated 1050–1080 universe of compounds (Martin, 1997). In addition, most conventional assays are limited to screening libraries of compounds against proteins with known function, excluding the large number of targets becoming available from genomics research.

Recently, a number of NMR-based screening methods have been employed to identify and design lead ligands for protein targets (see Table 1). These NMR-based strategies can augment ongoing conventional HTS for identifying leads and can be used to aid in lead optimization. All of these techniques take advantage of the fact that upon complex formation between a target molecule and a ligand, significant perturbations can be observed in NMR-sensitive parameters of either the target or the ligand. These perturbations can be used qualitatively to detect ligand binding or quantitatively to assess the strength of the binding interaction. In addition, some of the techniques allow the identification of the ligand binding site or which part of the ligand is responsible for interacting with the target. In this article, the current state of NMR-based screening is reviewed.

1. How do ribozymes work? 241

2. The hammerhead RNA as a prototype ribozyme 242

2.1 RNA enzymes 242

2.2 Satellite self-cleaving RNAs 242

2.3 Hammerhead RNAs and hammerhead ribozymes 244

3. The chemical mechanism of hammerhead RNA self-cleavage 246

3.1 Phosphodiester isomerization via an SN2(P) reaction 247

3.2 The canonical role of divalent metal ions in the hammerhead ribozyme reaction 251

3.3 The hammerhead ribozyme does not actually require metal ions for catalysis 254

3.4 Hammerhead RNA enzyme kinetics 257

4. Sequence requirements for hammerhead RNA self-cleavage 260

4.1 The conserved core, mutagenesis and functional group modifications 260

4.2 Ground-state vs. transition-state effects 261

5. The three-dimensional structure of the hammerhead ribozyme 262

5.1 Enzyme–inhibitor complexes 262

5.2 Enzyme–substrate complex in the initial state 264

5.3 Hammerhead ribozyme self-cleavage in the crystal 264

5.4 The requirement for a conformational change 265

5.5 Capture of conformational intermediates using crystallographic freeze-trapping 266

5.6 The structure of a hammerhead ribozyme ‘early’ conformational intermediate 267

5.7 The structure of a hammerhead ribozyme ‘later’ conformational intermediate 268

5.8 Is the conformational change pH dependent? 269

5.9 Isolating the structure of the cleavage product 271

5.10 Evidence for and against additional large-scale conformation changes 274

5.11 NMR spectroscopic studies of the hammerhead ribozyme 278

6. Concluding remarks 280

7. Acknowledgements 281

8. References 281

1. How do ribozymes work? 241

The discovery that RNA can be an enzyme (Guerrier-Takada et al. 1983; Zaug & Cech, 1986) has created the fundamental question of how RNA enzymes work. Before this discovery, it was generally assumed that proteins were the only biopolymers that had sufficient complexity and chemical heterogeneity to catalyze biochemical reactions. Clearly, RNA can adopt sufficiently complex tertiary structures that make catalysis possible. How does the three- dimensional structure of an RNA endow it with catalytic activity? What structural and functional principles are unique to RNA enzymes (or ribozymes), and what principles are so fundamental that they are shared with protein enzymes?